Chronic myeloid leukemia (CML) is a hematological stem cell disorder characterized by excessive proliferation of cells of the myeloid lineage. The hallmark of CML is the Philadelphia chromosome, which arises from a reciprocal translocation between chromosomes 9 and 22 (Rowley, 1973). The molecular consequence of this translocation is the replacement of the first exon of c-Abl with sequences from the Bcr gene resulting in a Bcr-Abl fusion gene whose protein product shows enhanced tyrosine kinase activity (Bartram, et al., 1983; Ben-Neriah, et al., 1986; Heisterkamp et al, 1983; Konopka, et al., 1984; Shtivelman et al., 1985). The Bcr-Abl oncoprotein in CML is a 210-kD protein that contains 902 or 927 amino acids of Bcr fused to the expression product of exons 2–11 of c-Abl (Ben-Neriah, et al., 1986; Shtivelman et al., 1985). Found in 95% of patients with CML, p210 Bcr-Abl is also present in approximately 5–10% of adults with acute leukemia for whom there is no evidence of antecedent CML (Kruzrock, et al., 1988). Another Bcr-Abl fusion protein of 185 kD containing Bcr sequences from exon 1 (426 amino acids) fused to exons 2–11 of c-Abl, occurs in 10% of adult cases and 5–10% of pediatric cases of acute lymphoblastic leukemia (ALL), but not in CML (Clark, et al., 1988; Hermans et al., 1987). It is believed that this single chromosomal rearrangement is sufficient to initiate the development of these diseases and may be the only molecular abnormality in early stage disease.
Protein kinases are a large family of homologous proteins comprising 2 major subfamilies, the protein serine/threonine kinases and protein tyrosine kinases (PTKs). Protein kinases function as components of signal transduction pathways, playing a central role in diverse biological processes such as control of cell growth, metabolism, differentiation, and apoptosis. The development of selective protein kinase inhibitors that can block or modulate diseases with abnormalities in these signaling pathways is considered a promising approach for drug development. However, due to the structural and functional similarities of kinases, it is virtually impossible to make an inhibitor specific to a single kinase. Because of their deregulation in human cancers, Bcr-Abl, epidermal growth factor receptor (EGFR), HER2, and protein kinase C (PKC), were among the first protein kinases considered as targets for the development of selective inhibitors. As protein kinases have been implicated in more human cancers (Kolibaba and Drucker, 1997), drug-discovery efforts have been extended and several first-generation small-molecule inhibitors are now in various stages of development. A selection of these agents is shown in FIG. 1 (Drucker and Lyndon, 2000).
A kinase inhibitor, STI-571 (4-[(4-methyl-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyrodnyl)-2-pyrimidinyl]amin]phenyl]benzamide methanesul-fonate; Glivec®, Novartis, Basel, Switzerland) was initially identified in a screen for inhibitors of the platelet derived growth factor receptor (PDGFR) (Buchdunger et al., 1995). This ATP analog of the class 2-phenylaminopyrimidine, was found to have some selectivity for specific kinases including cdc2/cyclin B, c-FGR, protein kinase C γ and v-Abl. As the constitutive activation of v-Abl is believed to be sufficient for the development of CML, it was seen as an ideal target for validating the clinical utility of protein kinase inhibitors in the treatment of cancer.
The Abl oncogene was isolated originally from the genome of the Abelson murine leukemia virus (A-MuLV) (Rosenberg and Witte, 1988). This acutely transforming replication-defective virus encodes a transforming protein (p160v-Abl) with tyrosine-specific protein kinase activity. A-MuLV transforms fibroblasts in vitro and lymphoid cells in vitro and in vivo and was formed by recombination between Moloney murine leukemia virus (M-MuLV) and the murine c-Abl gene (Rosenberg and Witte, 1988).
As with many of the viral oncogenes, there are cellular equivalents that are involved in growth and differentiation. c-Abl is expressed normally in cells where it shuttles between the cytoplasm and the nucleus. This shuttling is driven by its three nuclear localization sequences (NLS) and one nuclear export sequence (NES). The NES is located in the C-terminus of c-Abl and contains a characteristic leucine rich motif. Mutation of a single leucine (L1064A) is sufficient to disrupt the function of the NES and result in the nuclear localization of c-Abl (Taagepera et al., 1998). Alternatively, the export of wild type c-Abl from the nucleus may be inhibited by the drug leptomycin B (LMB; NSC-364372D; PD114,720; elactocin), which functions by inactivating the nuclear export mediator CRM-1/exportin-1. The inactivation of the CRM-1 is irreversible and export is arrested until new protein is synthesized.
Cell adhesion and intracellular localization of c-Abl control the kinetics of its activation. c-Abl kinase activity is dependent on integrin mediated cell adhesion (Lewis et al, 1996). Upon cell adhesion, the cytoplasmic pool of c-Abl is reactivated within 5 minutes; however, the nuclear pool of c-Abl does not become reactivated for about 30 minutes, suggesting that activation occurs in the cytoplasm and the activated protein is translocated into the nucleus. In quiescent and G1 cells, nuclear c-Abl is kept in an inactive state by the nuclear retinoblastoma protein (RB) that binds to the c-Abl tyrosine kinase domain and inhibits its activity. Phosphorylation of RB by the cyclin dependent kinases (CDKs) at the G1/S boundary disrupts the RB-c-Abl complex, leading to activation of the c-Abl kinase. Nuclear c-Abl is part of the RB-E2F complex; thus, the G1/S activation of c-Abl likely contributes to the regulation of genes involved in S-phase entry.
The nuclear c-Abl can be activated by DNA damage (Baskaran et al., 1997) to induce the p73 protein, which is a functional homolog of the tumor suppressor p53 (Gong, et al., 1999; Jost et al., 1997; Wang, 2000). Overexpression of p73 can activate the transcription of p53-responsive genes and inhibit cell growth in a p53-like manner by inducing apoptosis (Jost et al., 1997). Unlike c-Abl, Bcr-Abl is constitutively activated and is localized almost exclusively to the cytoplasm. The Bcr-Abl kinase activates a number of signal transduction pathways involved in cell proliferation and apoptosis (Warmuth, M., et al. 1999). For example, Bcr-Abl, which is typically cytoplasmic, can abrogate the dependence on interleukin-3 in hematopoietic cell lines (Daley et al., 1992) and the dependence on adhesion in fibroblastic cells (Renshaw et al., 1995). In addition, cytoplasmic Bcr-Abl can inhibit apoptosis through the activation of PI3-kinase, Akt and other mechanisms (Skorski et al., 1997; Amarante-Mendes et al., 1998). This mislocalization may be essential to the pathology of the protein.
STI 571 was shown to suppress the proliferation of Bcr-Abl-expressing cells in vitro and in vivo (Jost et al., 1999). In colony-forming assays of peripheral blood or bone marrow from patients with CML, STI 571 caused a 92–98% decrease in the number of Bcr-Abl colonies formed, with minimal inhibition of normal colony formation. (Jost et al., 1999) However, continual suppression of the Bcr-Abl tyrosine kinase is required for maximal clinical benefit in CML. Early studies demonstrated that Bcr-Abl-expressing cells could be rescued from apoptotic cell death if STI 571 were washed out of the cells within 16 hours of initial exposure (Drucker et al., 1996). If a tyrosine kinase inhibitor inhibited proliferation of Bcr-Abl-positive cells without inducing cell death, then long-term therapy would likely be required, suggesting that a well-tolerated, oral formulation of the drug would be needed.
Early pharmacokinetic studies in rats and dogs demonstrated that bioactive concentrations of STI571 are readily achieved in the circulation. However, the half-life of the drug is relatively short, 12–14 hours. In vivo STI571 treatment of nude mice injected with human leukemic cells was shown to eradicate 70–100% of the tumors. However, maintenance of a sufficiently high level of drug was essential for effective treatment. Mice receiving drug one or two times per day did not show significant improvements, whereas the mice receiving drug three times per day greatly improved (le Coutre et al., 1999). Additionally, relapse was seen in some animals which seemed to be mainly dependent on initial tumor load.
These pharmacokinetic properties make the drug less desirable for long term use. If a patient must take the drug long term, the chances of a patient maintaining a strict regimen of taking the drug on a strict interval decrease. No maximum tolerated dose has been identified for STI571 (Drucker, 2001a); however, its side effects are non-trivial for a drug that one must take in a regimented manner for an indefinite period of time. The most common side effects include nausea (in 43 percent of patients), myalgias (41 percent), edema (39 percent) and diarrhea (25 percent). Other side effects included fatigue, rash, dyspepsia, vomiting, theormbocytopenia, neutropenia and arthralgias.
Fluctuations in drug levels allow for the development of drug resistance which has been shown to happen in tissue culture (le Coutre et a., 2000). Treatment of LAMA84 human bcr-abl cells in culture with an initially sub-lethal, but continuously increasing dose of STI571 resulted in the development of a resistant cell line that was ten-fold more resistant to STI571 as compared to the parental cell line. Effectively, the process would be mimicked in a patient that did not adhere to a proper drug regimen. The cancer cells would be exposed to an insufficient dose of drug to induce quiescence or kill the more malignant cancer cells, allowing expansion of the resistant cells. Resistance develops by the amplification of the bcr-abl gene, a process which occurs during the blast crisis of the disease. Lack of adherence to the proper dosing regimen could hasten the onset of the blast crisis stage of the disease by promoting amplification of the gene.
Patients not responsive to standard interferon-α therapy were enrolled on a study to test the efficacy of STI571 as a chemotherapeutic agent (Drucker et al., 2001a). The study demonstrated that a relatively high level of drug must be maintained to have a therapeutic effect. Responses to STI571 were evaluated in two ways, hematologic, with complete response defined as a white-cell count of less than 10,000 per cubic millimeter; and cytogenetic, with a major response defined as less than 35 percent of the 20 metaphase cells analyzed positive for the Philadelphia chromosome. Hematologic responses were seen within two weeks, and all but one patient treated with 300 mg or more per day showed complete hematological response within four weeks. Only 31 percent of the patients had major cytogenetic responses, including 7 percent that showed complete cytogenetic remission. Cytogenetic responses occurred as early as two months and as late as 10 months. However, blood counts were maintained within normal limits regardless of whether a cytogenetic response was observed. Additionally, during treatment with STI571, blood counts gradually returned to normal during the first month, suggesting that the drug does not rapidly induce apoptosis, as would be expected with standard chemotherapy. Therefore, the drug seems to act by holding the diseased cells in abeyance rather than by killing them. This further emphasizes the need to maintain patients on STI571 indefinitely.
STI571 was also tested for efficacy in the treatment of patients who had entered the blast crisis of CML and acute lymphoblastic leukemia (ALL) (Drucker et al., 2001b). The blast crisis is highly refractory to treatment. The rate of response to standard induction chemotherapy is approximately 20 percent in myeloid blast crisis and 50 percent in lymphoid blast crisis. However, the remission seen is typically short lived. The blast crisis phase of both diseases is associated with genetic instability and multiple genetic abnormalities. Of the 58 patients enrolled in the study, 16 died due to disease progression. Other serious adverse events included nausea and vomiting, febrile neutropenia, elevated liver enzyme levels, exfoliative dermatitis, gastric hemorrage, renal failure, pancytopenia and congestive heart failure. The improvements seen in some of the patients were short lived. Of the 38 patients with myeloid blast crisis, 4 had complete hematologic remission and 17 had a decrease in blasts in the marrow to less than 15 percent. Of these 21 that showed improvement, 9 subsequently relapsed between 42 and 194 days (median 84), 7 remained in remission with a follow-up of 101 to 349 days and three discontinued the study due to adverse events. Of the 14 patients with lymphoid blast crisis who had a response to STI571, 12 relapsed between 42 and 123 days (median 58). One patient remained in remission for 243 days of follow-up. The authors of the study stated that although Bcr-Abl plays a role in the blast crisis, inhibition of the single protein alone is insufficient to treat the disease and that combination with a second agent is required.